Anti rap1

Nuclear Cytosolic Fractionation Kit (BioVision) was used to obtain protein extracts. Protein concentration was determined using the Bio‐Rad DC Protein Assay (Bio‐Rad). Up to twenty micrograms of nuclear protein extracts was separated in SDS–polyacrylamide gels by electrophoresis. After protein transfer onto nitrocellulose membrane, the membranes were incubated with the indicated antibodies. Antibody binding was detected after incubation with a secondary antibody coupled to horseradish peroxidase using chemiluminescence with ECL detection KIT (GE Healthcare).
Primary antibodies: anti‐TRF1 1:1,000 (BED5, Bio‐Rad), anti‐TRF1 1:500 (homemade), anti‐SMC‐1 1:2,000 (Bethyl), anti‐AKT1 1:500 (Millipore), anti‐p‐AKT 1:500 (Ser473, Cell Signaling Cell Signaling Technology), anti‐S6 1:500 (Cell Signaling Cell Signaling Technology), anti‐p‐S6 1:500 (Ser240/244, Cell Signaling Technology), anti‐TIN2 1:1,000 (Abcam), anti‐RAP1 1:1,000 (Bethyl).
For the quantification, protein‐band intensities have been quantified by densitometric analysis with ImageJ software. The Trf1 total levels have been normalized versus SMC1 and the mean of the Trf1/SMC1 ratio deriving from at least 3 different replicates has been used to generate the chart.

For immunofluorescence analyses, we plated the cells in a proper density in cell culture μCLEAR plates (Greiner) and we fixed them in 4% formaldehyde in PBS. We incubated the cells with 0.25% Triton in PBS followed by 5% BSA in PBS.
Tissue sections were fixed in 10% buffered formalin (Sigma) and embedded in paraffin. After deparaffinization and citrate antigen retrieval, we incubated the slides with 0.5% Triton in PBS and blocked them with 1% BSA and 10% Australian FBS (Genycell) in PBS.
The antibodies were applied overnight in antibody diluents with background reducing agents (Invitrogen).
Primary antibodies: anti‐Rap1 1:500 (BL735, Bethyl), anti‐γH2AX Ser139 1:500 (05‐636, Millipore), anti‐TRF1 1:500 (BED5, Bio‐Rad).
Images were obtained using a confocal ultraspectral microscope (Leica TCS‐SP5). Quantifications were performed with Definiens software.

Total protein extracts were obtained using RIPA extraction buffer and protein concentration was determined using the Bio-Rad DC Protein Assay (Bio-Rad). Up to 20 micrograms of protein per extract were separated in SDS–polyacrylamide gels by electrophoresis. After protein transfer onto nitrocellulose membrane (Whatman), the membranes were incubated with the indicated antibodies. Antibody binding was detected after incubation with a secondary antibody coupled to horseradish peroxidase using chemiluminescence with ECL detection KIT (GE Healthcare). Primary antibodies: anti-TRF1 (BED5, Cell Signaling), anti-RAP1 (Bethyl), anti-SMC-1 (Bethyl). Quantifications: Protein-band intensities were measured with ImageJ software and normalized against the loading control.